Correlation between thermostability and stability of glycosidases in ionic liquid.

The activity and stability of a β-glycosidase (Thermus thermophilus) and two α-galactosidases (Thermotoga maritima and Bacillus stearothermophilus) were studied in different hydrophilic ionic liquid (IL)/water ratios. For the ILs used, the glycosidases showed the best stability and activity in 1,3-dimethylimidazolium methyl sulfate [MMIM][MeSO(4)] and 1,2,3-trimethylimidazolium methyl sulfate [TMIM][MeSO(4)]. A close correlation was observed between the thermostability of the enzymes and their stability in IL media.

Rational design of a GH1 beta-glycosidase to prevent self-condensation during the transglycosylation reaction.

Mutant N282T of a thermostable beta-glycosidase from GH1 family (TtbetaGly) presenting a high transglycosidase activity was previously obtained by directed evolution. However, it displays a self-condensation activity with the donor 2-nitrophenyl-beta-d-galactopyranoside (oNPGal) which competes with the condensation reaction and entails undesirable effects. In order to prevent this reaction, we rationally modified this enzyme at the [+1]/[+2] subsites so that oNPGal would bind less tightly.

Evolved beta-galactosidases from Geobacillus stearothermophilus with improved transgalactosylation yield for galacto-oligosaccharide production.

A mutagenesis approach was applied to the beta-galactosidase BgaB from Geobacillus stearothermophilus KVE39 in order to improve its enzymatic transglycosylation of lactose into oligosaccharides. A simple screening strategy, which was based on the reduction of the hydrolysis of a potential transglycosylation product (lactosucrose), provided mutant enzymes possessing improved synthetic properties for the autocondensation product from nitrophenyl-galactoside and galacto-oligosaccharides (GOS) from lactose. The effects of the mutations on enzyme activity and kinetics were determined.

Directed evolution of the alpha-L-fucosidase from Thermotoga maritima into an alpha-L-transfucosidase.

The alpha-L-fucosidase from Thermotoga maritima (Tm alpha fuc) was converted into alpha-L-transfucosidase variants by directed evolution. The wild-type enzyme catalyzes oligosaccharide synthesis by transfer of a fucosyl residue from a pNP-fucoside donor to pNP-fucoside (self-condensation) with alpha-(1-->3) regioselectivity or pNP-galactoside (transglycosylation) with alpha-(1-->2) regioselectivity at low yields (7%). The wild-type enzyme was submitted to one cycle of mutagenesis, followed by rational recombination of the selected mutations, which allowed identification of variants with improved transferase activity.